Biochimica et Biophysica A cta, 1171 (1992) 201-204
201
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90430
Short Sequence-Paper
Cloning and sequencing of a cDNA for the 8-subunit of photosynthetic ATP-synthase (EC 3.6.1.34) from pea
(Pisum sativum) Jan A. Hoesche and Richard J. Berzborn Department of Biology, Lehrstuhl Biochemie der Pflanzen, Ruhr- Universitiit Bochum, Bochum (Germany)
(Received 28 September 1992)
Key words: Photophosphorylation; Reconstitution; Antibody; (P. sativum) hgtl0 cDNA clones for the nuclear encoded subunit 6 of chloroplast ATP-synthase from Pisum sativum have been isolated. The 5' end was completed by PCR. The sequenced cDNA codes for the import precursor. N-Terminal sequencing of the mature protein isolated from chloroplasts revealed that the processing sites of the transit peptide from Pisum sativum and Spinacea oleracea are similar. The overall homology of the deduced amino acid sequences of the mature S proteins from higher plants is about 40%. The conservation among hydrophilic residues is higher than for hydrophobic ones, indicating that the surface of ~5 is important for its function within the ATP-synthase. The ATP-synthase of chloroplasts uses the energy of protons, p u m p e d during illumination into the thylakoid lumen, to catalyze the photosynthetic A T P production. The 6 subunit of the peripheral subcomplex CF 1 (a3,~3,y,c~,E [1]), considered to be a mere connecting protein [2] between CF 1 and the m e m b r a n e integral CF0, seems not to be necessary for rebinding of CF~, but to play an unknown functional role [3]. It is able to bind to CF 0 and to block the proton effiux from thylakoids after removal of CF 1 by E D T A [4]. The C-terminus of 6 is exposed in situ and, significantly, antibodies against 8 inhibit photophosphorylation [5]. Thus, S probably is involved in the transduction of the energy of the proton motive force across the thylakoid m e m b r a n e to the active sites on CF 1. Since CFa S has direct contact to CF 0 I [6], protons or a conformational wave could proceed via the S subunit at the interphase between CF 0 and CF 1 [7,8]. We have produced monoclonal antibodies against an epitope on the binding surface of Spinacea oleracea CF~ 6 towards CF 0 (W. Finke and R.J. Berzborn, unpublished data); they crossreact with 6 from Pisum
sativum, not however with S from Zea mays. Also with
polyclonal antisera there is an immunological crossreaction between the 6 polypeptides of S. oleracea and P. sativum, but not with 6 from Z. mays. However, hybrid reconstitution of photophosphorylation with E D T A treated thylakoids and CF~, including 6, from different species is possible. With the coupling factors of spinach and Escherichia coli the hybrid reconstitution is only structural, i.e., after plugging of H + leaks residual ATP-synthase can be energized again [9]. Hybrid reconstitution between spinach and pea or maize leads in addition to catalytic activity of the readded enzymes [10]. To understand the interactions within the ATP-synthase complex and the role of subunit 6 in more detail, we want to elucidate conserved structural and functional elements on CF 1 8 of higher plants. Therefore, we are cloning and sequencing this nuclear encoded subunit from P. sativum, Sorghum bicolor and Z. mays, to compare them with the known sequence from S. oleracea and with the corresponding, homologous proteins from bacteria and mitochondria. In this publication we report our results on the CF~ subunit 6 of P. sativum.
Correspondence to: R.J. Berzborn, Biochemie der Pflanzen, Abt. Biologie, Ruhr-Universit~it, Box 102148, 4630 Bochum l, Germany. The reported nucleotide sequence data have been deposited at the GenBank/EMBL Nucleotide Sequence databases under accession number M94558. Abbreviations: RACE-PCR, rapid amplification of cDNA ends polymerase chain reaction; OSCP, oligomycin sensitivity confering protein of mitochondrial ATPase.
Restriction endonucleases, DNA-modifying enzymes and radiolabeled nucleotides were purchased. Oligonucleotides were synthesized on a Pharmacia Gene Assembler Plus synthesizer by the phosphoamidite method (in collaboration with the D e p a r t m e n t of Biochemistry, Faculty of Chemistry, Ruhr-Universit~it Bochum).
202 of S. oleracea [11] (a gift of Prof. Dr. R.G. Herrmann, Mfinchen). For RACE-PCR [12] on mRNA from 2-week-old P. satit'um seedlings, oligos were used corresponding to
A Agtl0 cDNA library of P. sativum, (a gift of Prof. Dr. U. Fliigge, Wfirzburg) was screened using a 630 bp 32p-labeled E c o R I / E c o R I fragment from pSocg-4E1 encoding most of the mature protein of the ~ subunit 1 49
ACC AAT CAC AAC ACA AAC TAA ACA CCC TCA TTT CAT TTC ACT ACA ACT ATT TCT TAC CTC ACC CTT CTT CTT CCT CTT CTT CCT
1 97
CCC ATA
M CCA ACA ATG Rb.
13 145
S K TCC AAA
H I CAC ATC
29 193
L N CTC AAC
L CTC
S S TCC TCT
45 241
L K CTC AAA
L CTC
P L T K T CCC CTC ACC AAA ACC
61 289
G A GGA GCC
R M $ S S L A A G S AGA ATG TCA TCC CTA GCC GCC GGC AGC
77 337
A D GCC GAC
L A N TTA GCA AAT
S N TCA AAC
93 385
F D TTC GAC
K I E AAA ATC GAA
Q CAA
109 433
F TTC
S S TCC AGC
125 481
E GAG
F A T TTC GCC ACT
T ACC
S G TCA GGC
141 529
N AAC
V GTC
L I TTG ATC
D GAC
S K R I TCG AAG AGG ATC
157 577
K E AAG GAG
F E TTT GAA
F V TTT GTT
173
V
V
T
S L Q H T T A TCT CTA CAG CAC ACC ACC GCT
P K T T N I CCC AAA ACC ACA AAC ATC
P I CCA ATA
S
A GCG
V
S T TCA ACC
28 192
F Y S P K TTC TAC TCC CCA AAA
L K CTC AAA
L K CTC AAA
44 240
R R S T G G A CGC CGC TCC ACC GGC GGA GCC
N T AAT ACC
L D A I T CTC GAC GCA ATA ACC P K V CCC AAG GTG
S T K TCC ACC AAA
R Q CGC CAA
F Q P H T TTC CAA CCA CAC ACA
L
E
L TTA
76 336
A D GCC GAC
92 384
Y TAT
108 432
L I G CTT ATA GGA
124 480
F TTC
L CTT
140 528
D M I I D I GAT ATG ATC ATC GAC ATT
I ATT
156 576
L V CTT GTG
172 624
L T D TTA ACT GAT S
H
H
T E ACA GAG L
ACT
189 673
A K GCA AAA
Q V CAG GTT
Q K CAG AAG
205 721
T ACT
L L D CTT CTT GAT
P S CCA AGT
221 769
N AAC
T G ACT GGC
237 817
E I A GAA ATT GCT
865 913 961
GCA TTC TTC GTG CTT CCT TTC AAT TTG GTT TAC TCA AAT TTT TTG GAT TAT AAG TAT ATT TAT GTA TCT ATG TAT GAA GTG TAC AAC CTT CTT ACA ATA ATC ATA TTA ATA TTG AGT PA TTA TAA AAA AAA AAA AA
1009
60 288
F D TTC GAC
H N CAC AAC
GTT GTG
A GCT
L CTC
Y A A A TAC GCA GCC GCT
625
S K TCT AAG
12 144
P I CCC ATC
Y N T TAC AAT ACT K
H CAC
R K CGA AAA
V E D GTC GAA GAT
V
S L TCA CTC
A
Q
I
~CA ¢~ CAT TTG GCT CAG ATT Amp. L T G A K K V R T K CTT ACT GGG GCT AAG AAA GTG AGG ACT AAA RT L V A G F T V R Y G TTG GTG GCT GGT TTT ACT GTC AGG TAT GGG
F I TTT ATT
Q I D CAA ATT GAT
48 96
L T CTC ACT
L F S D CTT TTC TCT GAC
TCG GTT GTG AAG
CAC CAC CCT CTA
TTG GAg
D M S V K R K GAT ATG AGT GTT AAG AGG AAA L G D I TTG GGT GAT ATC
L E CTC GAG
Q L A V CAA CTT GCT GTA
188 672 204 720 220 768 236 816
* TAA
251 864
TGT AAT TTT TCC TCA AGT CCC TGC TTC
912 960 1008 1025
Fig. 1. c D N A nucleotide sequence and deduced amino acid sequence of the complete precursor of the CF 1 6 subunit from Pisum sati~um. The arrow indicates the beginning of the mature protein. The sequence parts that correspond to the oligo nucleotide primers used in the R A C E - P C R (RT = reverse transcription, A m p = amplification), the ribosome binding site (bp 105-113, Rb.) and the potential polyadenylation signal (bp 978 to 982, PA) are underlined.
203 bps 699-715 (reverse transcription) and bps 648-665 (amplification) of the cDNA of the complete precursor of the 6 subunit from P. sativum. The cDNA was partially digested with AluI, cloned into M13 mp18/19 [13] and sequenced [14] using Taq DNA polymerase and 7'-deaza-dGTP (Promega) at 72°C. 30 mg of isolated CF 1 [15] from P. sativum was separated on 12.5% (w/v) polyacrylamide gels [16]. The 6 band, positive in Western blots with antisera against spinach CF~ 6, was cut out, purified on a second SDS-PAGE (18% w/v) and transferred to a PVDF membrane (Millipore). Automated Edman degradation was done on an Applied Biosystems 477 A Gasphase sequenator [17] in collaboration with Dr. H.E. Meyer, faculty of medicine, Ruhr-Universit~it Bochum, Germany). Screening of a hgtl0 cDNA library of P. sativum led to three different cDNA clones, approx. 500, 700 and 900 bp in length, all containing 5' truncated forms of cDNA for the 6 subunit. From the sequence of this cDNA the amino acid sequence of the mature protein and part of a transit peptide was deduced. The missing 5' end could be determined by RACE-PCR; a 650 bp fragment was amplified, subcloned and sequenced. The combined complete cDNA sequence is shown in Fig. 1. The cDNA consists of 1025 bp, including 13 bp of the poly(A) tail. An open reading frame of 753 nucleotides codes for the 251 amino acid precursor (27 624 Da). In addition the clone encodes 108 bp of 5' untranslated sequence with a potential ribosome binding site at bp 105 to 113 and 164 bp of 3' untranslated sequence with a potential polyadenylation site at bp 978 to 982 (Fig. 1).
To localize the processing site of the import precursor, the 6 subunit of P. sativum CF 1 was isolated from chloroplasts and sequenced. The mature protein starts with SSLA .... (bold in Fig. 2); a transit peptide of 64 amino acids has been cleaved off. The mature 6 subunit consists of 187 amino acids (20598 Da). Comparison of the deduced amino acid sequence of the 6 subunit of P. sativum with the sequence of Sorghum bicolor, Z. mays and S. oleracea [11,18], showed that 40% (75 amino acids) of the amino acids of the mature proteins are identical; including conservative exchanges 85% are homologous (data not shown). In spite of immunological differences the isolated proteins and genes belong to the same protein family, the subunits 6 of the H ÷ translocating F0F 1 ATPases. The transit peptides of the 6 subunits from higher plants are similar in length (60-70 amino acids). Allthough only 10 amino acids are conserved, the peptides display the characteristics of stromal transit peptides [19], i.e., they are rich in serine, threonine, lysine and argine and contain nearly no acidic residues. Close to the processing site positive charges are found, near position - 2 and around position -10, a requirement claimed for the processing proteinase [19] (cp. Fig. 2). Comparison of the 6 sequences of higher plants with the sequences of the corresponding proteins from mitochondria (OSCP, not the socalled 6 subunit) and bacteria (21 species available, data not shown) reveals that only 5 amino acids (2.8%, marked by a ( + ) in Fig. 2) are strictly conserved among all species [18]. In addition only 22 conservative exchanges occurred (marked by a ( ~ ) in Fig. 2). However, these few strictly conserved and conservatively exchanged amino acids are clustered near the N-termini, the middle part and
P.
sat.
MASLQHTTASLHSKHIPKTTNILTR
S.
ole.
MAALQNPVA.
E.
coli
..................................................................
P. S. E.
sat. ole. coli
LGARMSSLAAGSYAAALADLANSNNTLDAITADFDKIE.
P.
sat.
EF.ATTSGFQPHTHNFLNVLIDSKRIDMIIDIIKEFEFVY.NTLTDTELVVVTSVVKLESHHLAQI
S.
ole.
EI. I T T S G L Q P H T A N F I N I L I D S E R I N L V K E I L N E F E D V F .
E.
coli
.A V C G E . Q L D E N G Q N L I R V M A E N G R L N A L P D V L E Q F
...... KPI. LNLSSSTFYSPKLKLKLKLPLTKTRRSTGGA
LQSRTTTAVAALSTSSTTSPPKPFSLSFSSSTATFNPLRLKILTASKLTAKPRGGA
QLFSDPKVFDYFSSPIVEDSTKRQLIG
LGTRMVDSTASRYASALADVADVTOTLEATNSDVEKL .. M S E F I T V A R P Y A K A A F D F A V E H Q S V E R W Q . + + -
I. R I F S E E P V Y Y F F A N P V I S I D N K R S V L D
DMLAFAAEVTKNEQMAELLSGALAPETLAE _ _
S F I.
aaaaaaaaaaaaa
.
P.
sat.
S. E.
ole. coli
.
.
.
.
+
--
NKITGTEVAVVTSVVKLENDHLAQI
IHL. RAVSEATAEVDV
ISAAALSEQQLAK .
i --
BBBBBBB BBBBBBBB aaaaaaaaaaaaaaaaa AKQVQK... LTGAKKVRTKTLLDPSLVAGFTVRYGNTGSKFIDMSVKRKLEEIAAQIDLGDIQLAV AKGVQK... ITGAKNVRIKTVIDPSLVAGFTIRYGNEGSKLVDMSVKKQLEEIAAQLEMDDVTLAV SAAMEK... RLS. RKVKLNCKIDKSVMAGVI IRAG... DMVIDGSVRGRLERLADVLQS ....... . . . . . . + + -
Fig. 2. Alignment of the deduced amino acid sequences of the 6 subunits from P. satit,um (P. sat.) with the corresponding subunits of Spinacea oleracea (S. ole.) [11] and Escherichia coli (E. coli) [20], using the program clustal; three species out of the alignment of 21 sequences [18] are shown. The five amino acids conserved among all ~ subunits from plants, bacteria and the homologous mitochondrial proteins are marked by ( + ), conservatively exchanged by ( ~ ). The sequence areas that are predicted to form amphipathic a helices or the putative amphipathic/3 sheet are marked by ( a ) or (/3). The sequenced N-terminal residues of the isolated mature proteins are given in bold.
204 the C - t e r m i n i of the m a t u r e proteins. By s e c o n d a r y s t r u c t u r e p r e d i c t i o n a l g o r i t h m s and helical w h e e l analysis a m p h i p a t h i c a helices are p r e d i c t e d for the Ntermini, the m i d d l e p a r t a n d the C - t e r m i n i of the subunits of most species. A n a n t i p a r a l l e l a m p h i p a t h i c /3 s h e e t is also p r e d i c t e d n e a r l y in all 21 species [18]. Except for the five c o n s e r v e d residues, the s e c o n d a r y s t r u c t u r e of the C F 1 s u b u n i t 6 from h i g h e r p l a n t s a n d of the h o m o l o g o u s p r o t e i n s from m i t o c h o n d r i a a n d b a c t e r i a s e e m s to be m o r e i m p o r t a n t for the function than invariant r e s i d u e s at c e r t a i n positions in the sequence. T h e new s e q u e n c e of the m a t u r e CF~ s u b u n i t ,~ of P. s a t i v u m was c o m p a r e d in detail with the h o m o l o gous s e q u e n c e s from E. coil [20] a n d S. oleracea [11]. In the E. coli 6 s e q u e n c e 81 hydrophilic, 61 h y d r o p h o bic and 35 small a m i n o acids are found, in P. satit~urn CF~ ~ 96, 66 and 25, in S. oleracea 6 95, 68 a n d 24, respectively. Thus, on the a v e r a g e 10 (11) small r e s i d u e s have b e e n m u t a t e d to m o r e specific o n e s in the h i g h e r plant sequences, and the hydrophilicity of ~ has significantly increased. In spinach and p e a 6 18 of the r e m a i n i n g small r e s i d u e s are f o u n d in identical positions (Fig. 2). T h e total n u m b e r of i d e n t i t i e s in the 6 s e q u e n c e s b e t w e e n P. satiL'um and spinach is l a r g e r than b e t w e e n E. coli a n d spinach, as e x p e c t e d . In c o n n e c t i o n with hybrid r e c o n s t i t u t i o n (see above), the d e t a i l e d c o m p a r i s o n led to an a d d i t i o n a l interesting conclusion: the n u m b e r of h y d r o p h i l i c residues, identical in the ~ s e q u e n c e s of E. coli and spinach, is l a r g e r than that of identical h y d r o p h o b i c residues. This t e n d e n c y is i n c r e a s e d , if the n u m b e r a n d p r o p o r t i o n s of r e s i d u e s are c o u n t e d , which are identical in the r e s p e c t i v e g r o u p s b e t w e e n P. satiL~um a n d spinach 6 (Fig. 2 a n d T a b l e I). In c o n t r a s t to this, in families of soluble p r o t e i n s the h y d r o p h i l i c r e s i d u e s are usually less c o n s e r v e d t h a n the h y d r o p h o b i c ones in the protein core [21]. Since C F 1 ~ is a subunit of a p e r i p h e r a l m e m b r a n e p r o t e i n and soluble after isolation, most of its h y d r o p h o b i c r e s i d u e s should be l o c a t e d in the pro-
TABLE I Namber of identities in the primary structure of mature CFl subunit of Spinacea oleracea (187 residues), when compared with Pisum sativum CFI 6 (187 residues) and E. coil F1 ~ (177 residues)
Alignment as in Fig. 2. In brackets: % of identities in relation to all residues within the respective group of amino acids of 6 in the respective species. Comparison ofsubunits 6 from
Total identities
Hydrophilic DETSKR HNQCY
Hydrophobic LVIMFP W
Small AG
Spinach and
106
55 (57%of 96) 18 (22% of 81)
33 (50%of 66) 14 (23%of 61)
18 (72%of 25) 9 (26%of 35)
satil,um
Spinach and E. coli
41
tein core a n d most of the h y d r o p h i l i c r e s i d u e s at the surface. Thus, the surface of ~ is surprisingly well conserved. T h e function of ~ s e e m s to d e p e n d not only on its size a n d shape, b u t on its surface; its specific i n t e r a c t i o n s with o t h e r subunits of the A T P - s y n t h a s e c o m p l e x a r e most i m p o r t a n t . Using a n t i b o d i e s and limited proteolysis we have shown that C F 1 subunit in situ is mostly inaccessible, i n d e e d [22]. In hybrid r e c o n s t i t u t i o n the similarities on the surface of 6 from E. coli a n d S. oleracea w e r e not sufficient for catalytic r e c o n s t i t u t i o n [9]. W e p r o p o s e that the capacity of CFI subunit 6 to r e c o n s t i t u t e p h o s p h o r y l a t i o n activity, corr e l a t e s with the n u m b e r of identical h y d r o p h i l i c surface residues. F o r financial s u p p o r t we are i n d e b t e d to the Studie n s t i f t u n g des D e u t s c h e n V o l k e s (J.A.H.) and the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (Be 664 to R.J.B.).
References 1 Jagendorf, A.T., McCarty, R.E. and Robertson, D. (1991) in The Photosynthetic Apparatus: Molecular Biology and Operation (Bogorad, L. and Vasil, I.K., eds.), pp. 225-254, Academic Press, New York. 2 Smith, J.B. and Sternweis, P.C. (1977) Biochemistry 16, 306-311. 3 Xiao, J. and McCarty, R.E. (1989) Biochim. Biophys. Acta 976, 203-209. 4 Engelbrecht, S. and Junge, W. (1988) Eur. J. Biochem. 172, 213-218. 5 Berzborn, R.J. and Finke, W. (1989a) Z. Naturforsch. 44c, 153 160. 6 Beckers, G. Berzborn, R.J. and Strotmann, H. (1992) Biochim. Biophys. Acta 1101, 97-104. 7 Mitchell, P. (1985) FEBS Lett. 182, 1 7. 8 Boyer, P.D. (1987) Biochemistry 26, 8503-8507. 9 Engelbrecht, S., Deckers-Hebestreit, G., Altendorf, K.H. and Junge, W. (1989) Eur. J. Biochem. 181,485-491 10 Pucheu, N.L. and Berzborn, R.J. (1984) in Advances in Photosynthesis Res. (Sybesma, C., ed.), Vol. II, pp.571-574, M. Nijhoff/Dr. W. Junk Publ., The Hague. 11 Hermans, J., Rother, C., Steppuhn, J. and Herrmann, R.G. (1988) Plant Mol. Biol. 10, 323-330 12 Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Proc, Natl. Acad. Sci. USA 85, 8998-9002. 13 Yanisch-Perron, C., Viera, J. and Messing, J. (1985) Gene 33, 103-119. 14 Sanger, F.~ Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 15 Lien, S. and Racker, E. (1970) Methods Enzymol. 23, 547-555. 16 Lugtenberg, B., Meijers, J., Peters, R., Van der Hoek, P. and Van Alphen, L. (1975) FEBS Lett. 58, 254-258. 17 Hewick, R.M., Hunkapiller, N.W., Hood, L.E. and Dreyer, W.J. (1981) J. Biol. Chem. 256, 7990-7997. 18 Hoesche, J.A. and Berzborn, R.J. (1992) unpublished data. 19 Von Heijne, G., Steppuhn, J. and Herrmann, R.G. (1989) Eur. J. Biochem. 180, 535-545. 20 Walker, J.E., Saraste, M. and Gay, J.E. (1984) Biochim. Biophys. Acta 768, 164-200 21 Schulz, G.E. and Schirmer, R.H. (1979) Principles in protein structure, Springer, New York, p. 170 22 Berzborn, R.J. and Finke, W. (1989b) Z. Naturforsch. 44c, 480486.
201
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90430
Short Sequence-Paper
Cloning and sequencing of a cDNA for the 8-subunit of photosynthetic ATP-synthase (EC 3.6.1.34) from pea
(Pisum sativum) Jan A. Hoesche and Richard J. Berzborn Department of Biology, Lehrstuhl Biochemie der Pflanzen, Ruhr- Universitiit Bochum, Bochum (Germany)
(Received 28 September 1992)
Key words: Photophosphorylation; Reconstitution; Antibody; (P. sativum) hgtl0 cDNA clones for the nuclear encoded subunit 6 of chloroplast ATP-synthase from Pisum sativum have been isolated. The 5' end was completed by PCR. The sequenced cDNA codes for the import precursor. N-Terminal sequencing of the mature protein isolated from chloroplasts revealed that the processing sites of the transit peptide from Pisum sativum and Spinacea oleracea are similar. The overall homology of the deduced amino acid sequences of the mature S proteins from higher plants is about 40%. The conservation among hydrophilic residues is higher than for hydrophobic ones, indicating that the surface of ~5 is important for its function within the ATP-synthase. The ATP-synthase of chloroplasts uses the energy of protons, p u m p e d during illumination into the thylakoid lumen, to catalyze the photosynthetic A T P production. The 6 subunit of the peripheral subcomplex CF 1 (a3,~3,y,c~,E [1]), considered to be a mere connecting protein [2] between CF 1 and the m e m b r a n e integral CF0, seems not to be necessary for rebinding of CF~, but to play an unknown functional role [3]. It is able to bind to CF 0 and to block the proton effiux from thylakoids after removal of CF 1 by E D T A [4]. The C-terminus of 6 is exposed in situ and, significantly, antibodies against 8 inhibit photophosphorylation [5]. Thus, S probably is involved in the transduction of the energy of the proton motive force across the thylakoid m e m b r a n e to the active sites on CF 1. Since CFa S has direct contact to CF 0 I [6], protons or a conformational wave could proceed via the S subunit at the interphase between CF 0 and CF 1 [7,8]. We have produced monoclonal antibodies against an epitope on the binding surface of Spinacea oleracea CF~ 6 towards CF 0 (W. Finke and R.J. Berzborn, unpublished data); they crossreact with 6 from Pisum
sativum, not however with S from Zea mays. Also with
polyclonal antisera there is an immunological crossreaction between the 6 polypeptides of S. oleracea and P. sativum, but not with 6 from Z. mays. However, hybrid reconstitution of photophosphorylation with E D T A treated thylakoids and CF~, including 6, from different species is possible. With the coupling factors of spinach and Escherichia coli the hybrid reconstitution is only structural, i.e., after plugging of H + leaks residual ATP-synthase can be energized again [9]. Hybrid reconstitution between spinach and pea or maize leads in addition to catalytic activity of the readded enzymes [10]. To understand the interactions within the ATP-synthase complex and the role of subunit 6 in more detail, we want to elucidate conserved structural and functional elements on CF 1 8 of higher plants. Therefore, we are cloning and sequencing this nuclear encoded subunit from P. sativum, Sorghum bicolor and Z. mays, to compare them with the known sequence from S. oleracea and with the corresponding, homologous proteins from bacteria and mitochondria. In this publication we report our results on the CF~ subunit 6 of P. sativum.
Correspondence to: R.J. Berzborn, Biochemie der Pflanzen, Abt. Biologie, Ruhr-Universit~it, Box 102148, 4630 Bochum l, Germany. The reported nucleotide sequence data have been deposited at the GenBank/EMBL Nucleotide Sequence databases under accession number M94558. Abbreviations: RACE-PCR, rapid amplification of cDNA ends polymerase chain reaction; OSCP, oligomycin sensitivity confering protein of mitochondrial ATPase.
Restriction endonucleases, DNA-modifying enzymes and radiolabeled nucleotides were purchased. Oligonucleotides were synthesized on a Pharmacia Gene Assembler Plus synthesizer by the phosphoamidite method (in collaboration with the D e p a r t m e n t of Biochemistry, Faculty of Chemistry, Ruhr-Universit~it Bochum).
202 of S. oleracea [11] (a gift of Prof. Dr. R.G. Herrmann, Mfinchen). For RACE-PCR [12] on mRNA from 2-week-old P. satit'um seedlings, oligos were used corresponding to
A Agtl0 cDNA library of P. sativum, (a gift of Prof. Dr. U. Fliigge, Wfirzburg) was screened using a 630 bp 32p-labeled E c o R I / E c o R I fragment from pSocg-4E1 encoding most of the mature protein of the ~ subunit 1 49
ACC AAT CAC AAC ACA AAC TAA ACA CCC TCA TTT CAT TTC ACT ACA ACT ATT TCT TAC CTC ACC CTT CTT CTT CCT CTT CTT CCT
1 97
CCC ATA
M CCA ACA ATG Rb.
13 145
S K TCC AAA
H I CAC ATC
29 193
L N CTC AAC
L CTC
S S TCC TCT
45 241
L K CTC AAA
L CTC
P L T K T CCC CTC ACC AAA ACC
61 289
G A GGA GCC
R M $ S S L A A G S AGA ATG TCA TCC CTA GCC GCC GGC AGC
77 337
A D GCC GAC
L A N TTA GCA AAT
S N TCA AAC
93 385
F D TTC GAC
K I E AAA ATC GAA
Q CAA
109 433
F TTC
S S TCC AGC
125 481
E GAG
F A T TTC GCC ACT
T ACC
S G TCA GGC
141 529
N AAC
V GTC
L I TTG ATC
D GAC
S K R I TCG AAG AGG ATC
157 577
K E AAG GAG
F E TTT GAA
F V TTT GTT
173
V
V
T
S L Q H T T A TCT CTA CAG CAC ACC ACC GCT
P K T T N I CCC AAA ACC ACA AAC ATC
P I CCA ATA
S
A GCG
V
S T TCA ACC
28 192
F Y S P K TTC TAC TCC CCA AAA
L K CTC AAA
L K CTC AAA
44 240
R R S T G G A CGC CGC TCC ACC GGC GGA GCC
N T AAT ACC
L D A I T CTC GAC GCA ATA ACC P K V CCC AAG GTG
S T K TCC ACC AAA
R Q CGC CAA
F Q P H T TTC CAA CCA CAC ACA
L
E
L TTA
76 336
A D GCC GAC
92 384
Y TAT
108 432
L I G CTT ATA GGA
124 480
F TTC
L CTT
140 528
D M I I D I GAT ATG ATC ATC GAC ATT
I ATT
156 576
L V CTT GTG
172 624
L T D TTA ACT GAT S
H
H
T E ACA GAG L
ACT
189 673
A K GCA AAA
Q V CAG GTT
Q K CAG AAG
205 721
T ACT
L L D CTT CTT GAT
P S CCA AGT
221 769
N AAC
T G ACT GGC
237 817
E I A GAA ATT GCT
865 913 961
GCA TTC TTC GTG CTT CCT TTC AAT TTG GTT TAC TCA AAT TTT TTG GAT TAT AAG TAT ATT TAT GTA TCT ATG TAT GAA GTG TAC AAC CTT CTT ACA ATA ATC ATA TTA ATA TTG AGT PA TTA TAA AAA AAA AAA AA
1009
60 288
F D TTC GAC
H N CAC AAC
GTT GTG
A GCT
L CTC
Y A A A TAC GCA GCC GCT
625
S K TCT AAG
12 144
P I CCC ATC
Y N T TAC AAT ACT K
H CAC
R K CGA AAA
V E D GTC GAA GAT
V
S L TCA CTC
A
Q
I
~CA ¢~ CAT TTG GCT CAG ATT Amp. L T G A K K V R T K CTT ACT GGG GCT AAG AAA GTG AGG ACT AAA RT L V A G F T V R Y G TTG GTG GCT GGT TTT ACT GTC AGG TAT GGG
F I TTT ATT
Q I D CAA ATT GAT
48 96
L T CTC ACT
L F S D CTT TTC TCT GAC
TCG GTT GTG AAG
CAC CAC CCT CTA
TTG GAg
D M S V K R K GAT ATG AGT GTT AAG AGG AAA L G D I TTG GGT GAT ATC
L E CTC GAG
Q L A V CAA CTT GCT GTA
188 672 204 720 220 768 236 816
* TAA
251 864
TGT AAT TTT TCC TCA AGT CCC TGC TTC
912 960 1008 1025
Fig. 1. c D N A nucleotide sequence and deduced amino acid sequence of the complete precursor of the CF 1 6 subunit from Pisum sati~um. The arrow indicates the beginning of the mature protein. The sequence parts that correspond to the oligo nucleotide primers used in the R A C E - P C R (RT = reverse transcription, A m p = amplification), the ribosome binding site (bp 105-113, Rb.) and the potential polyadenylation signal (bp 978 to 982, PA) are underlined.
203 bps 699-715 (reverse transcription) and bps 648-665 (amplification) of the cDNA of the complete precursor of the 6 subunit from P. sativum. The cDNA was partially digested with AluI, cloned into M13 mp18/19 [13] and sequenced [14] using Taq DNA polymerase and 7'-deaza-dGTP (Promega) at 72°C. 30 mg of isolated CF 1 [15] from P. sativum was separated on 12.5% (w/v) polyacrylamide gels [16]. The 6 band, positive in Western blots with antisera against spinach CF~ 6, was cut out, purified on a second SDS-PAGE (18% w/v) and transferred to a PVDF membrane (Millipore). Automated Edman degradation was done on an Applied Biosystems 477 A Gasphase sequenator [17] in collaboration with Dr. H.E. Meyer, faculty of medicine, Ruhr-Universit~it Bochum, Germany). Screening of a hgtl0 cDNA library of P. sativum led to three different cDNA clones, approx. 500, 700 and 900 bp in length, all containing 5' truncated forms of cDNA for the 6 subunit. From the sequence of this cDNA the amino acid sequence of the mature protein and part of a transit peptide was deduced. The missing 5' end could be determined by RACE-PCR; a 650 bp fragment was amplified, subcloned and sequenced. The combined complete cDNA sequence is shown in Fig. 1. The cDNA consists of 1025 bp, including 13 bp of the poly(A) tail. An open reading frame of 753 nucleotides codes for the 251 amino acid precursor (27 624 Da). In addition the clone encodes 108 bp of 5' untranslated sequence with a potential ribosome binding site at bp 105 to 113 and 164 bp of 3' untranslated sequence with a potential polyadenylation site at bp 978 to 982 (Fig. 1).
To localize the processing site of the import precursor, the 6 subunit of P. sativum CF 1 was isolated from chloroplasts and sequenced. The mature protein starts with SSLA .... (bold in Fig. 2); a transit peptide of 64 amino acids has been cleaved off. The mature 6 subunit consists of 187 amino acids (20598 Da). Comparison of the deduced amino acid sequence of the 6 subunit of P. sativum with the sequence of Sorghum bicolor, Z. mays and S. oleracea [11,18], showed that 40% (75 amino acids) of the amino acids of the mature proteins are identical; including conservative exchanges 85% are homologous (data not shown). In spite of immunological differences the isolated proteins and genes belong to the same protein family, the subunits 6 of the H ÷ translocating F0F 1 ATPases. The transit peptides of the 6 subunits from higher plants are similar in length (60-70 amino acids). Allthough only 10 amino acids are conserved, the peptides display the characteristics of stromal transit peptides [19], i.e., they are rich in serine, threonine, lysine and argine and contain nearly no acidic residues. Close to the processing site positive charges are found, near position - 2 and around position -10, a requirement claimed for the processing proteinase [19] (cp. Fig. 2). Comparison of the 6 sequences of higher plants with the sequences of the corresponding proteins from mitochondria (OSCP, not the socalled 6 subunit) and bacteria (21 species available, data not shown) reveals that only 5 amino acids (2.8%, marked by a ( + ) in Fig. 2) are strictly conserved among all species [18]. In addition only 22 conservative exchanges occurred (marked by a ( ~ ) in Fig. 2). However, these few strictly conserved and conservatively exchanged amino acids are clustered near the N-termini, the middle part and
P.
sat.
MASLQHTTASLHSKHIPKTTNILTR
S.
ole.
MAALQNPVA.
E.
coli
..................................................................
P. S. E.
sat. ole. coli
LGARMSSLAAGSYAAALADLANSNNTLDAITADFDKIE.
P.
sat.
EF.ATTSGFQPHTHNFLNVLIDSKRIDMIIDIIKEFEFVY.NTLTDTELVVVTSVVKLESHHLAQI
S.
ole.
EI. I T T S G L Q P H T A N F I N I L I D S E R I N L V K E I L N E F E D V F .
E.
coli
.A V C G E . Q L D E N G Q N L I R V M A E N G R L N A L P D V L E Q F
...... KPI. LNLSSSTFYSPKLKLKLKLPLTKTRRSTGGA
LQSRTTTAVAALSTSSTTSPPKPFSLSFSSSTATFNPLRLKILTASKLTAKPRGGA
QLFSDPKVFDYFSSPIVEDSTKRQLIG
LGTRMVDSTASRYASALADVADVTOTLEATNSDVEKL .. M S E F I T V A R P Y A K A A F D F A V E H Q S V E R W Q . + + -
I. R I F S E E P V Y Y F F A N P V I S I D N K R S V L D
DMLAFAAEVTKNEQMAELLSGALAPETLAE _ _
S F I.
aaaaaaaaaaaaa
.
P.
sat.
S. E.
ole. coli
.
.
.
.
+
--
NKITGTEVAVVTSVVKLENDHLAQI
IHL. RAVSEATAEVDV
ISAAALSEQQLAK .
i --
BBBBBBB BBBBBBBB aaaaaaaaaaaaaaaaa AKQVQK... LTGAKKVRTKTLLDPSLVAGFTVRYGNTGSKFIDMSVKRKLEEIAAQIDLGDIQLAV AKGVQK... ITGAKNVRIKTVIDPSLVAGFTIRYGNEGSKLVDMSVKKQLEEIAAQLEMDDVTLAV SAAMEK... RLS. RKVKLNCKIDKSVMAGVI IRAG... DMVIDGSVRGRLERLADVLQS ....... . . . . . . + + -
Fig. 2. Alignment of the deduced amino acid sequences of the 6 subunits from P. satit,um (P. sat.) with the corresponding subunits of Spinacea oleracea (S. ole.) [11] and Escherichia coli (E. coli) [20], using the program clustal; three species out of the alignment of 21 sequences [18] are shown. The five amino acids conserved among all ~ subunits from plants, bacteria and the homologous mitochondrial proteins are marked by ( + ), conservatively exchanged by ( ~ ). The sequence areas that are predicted to form amphipathic a helices or the putative amphipathic/3 sheet are marked by ( a ) or (/3). The sequenced N-terminal residues of the isolated mature proteins are given in bold.
204 the C - t e r m i n i of the m a t u r e proteins. By s e c o n d a r y s t r u c t u r e p r e d i c t i o n a l g o r i t h m s and helical w h e e l analysis a m p h i p a t h i c a helices are p r e d i c t e d for the Ntermini, the m i d d l e p a r t a n d the C - t e r m i n i of the subunits of most species. A n a n t i p a r a l l e l a m p h i p a t h i c /3 s h e e t is also p r e d i c t e d n e a r l y in all 21 species [18]. Except for the five c o n s e r v e d residues, the s e c o n d a r y s t r u c t u r e of the C F 1 s u b u n i t 6 from h i g h e r p l a n t s a n d of the h o m o l o g o u s p r o t e i n s from m i t o c h o n d r i a a n d b a c t e r i a s e e m s to be m o r e i m p o r t a n t for the function than invariant r e s i d u e s at c e r t a i n positions in the sequence. T h e new s e q u e n c e of the m a t u r e CF~ s u b u n i t ,~ of P. s a t i v u m was c o m p a r e d in detail with the h o m o l o gous s e q u e n c e s from E. coil [20] a n d S. oleracea [11]. In the E. coli 6 s e q u e n c e 81 hydrophilic, 61 h y d r o p h o bic and 35 small a m i n o acids are found, in P. satit~urn CF~ ~ 96, 66 and 25, in S. oleracea 6 95, 68 a n d 24, respectively. Thus, on the a v e r a g e 10 (11) small r e s i d u e s have b e e n m u t a t e d to m o r e specific o n e s in the h i g h e r plant sequences, and the hydrophilicity of ~ has significantly increased. In spinach and p e a 6 18 of the r e m a i n i n g small r e s i d u e s are f o u n d in identical positions (Fig. 2). T h e total n u m b e r of i d e n t i t i e s in the 6 s e q u e n c e s b e t w e e n P. satiL'um and spinach is l a r g e r than b e t w e e n E. coli a n d spinach, as e x p e c t e d . In c o n n e c t i o n with hybrid r e c o n s t i t u t i o n (see above), the d e t a i l e d c o m p a r i s o n led to an a d d i t i o n a l interesting conclusion: the n u m b e r of h y d r o p h i l i c residues, identical in the ~ s e q u e n c e s of E. coli and spinach, is l a r g e r than that of identical h y d r o p h o b i c residues. This t e n d e n c y is i n c r e a s e d , if the n u m b e r a n d p r o p o r t i o n s of r e s i d u e s are c o u n t e d , which are identical in the r e s p e c t i v e g r o u p s b e t w e e n P. satiL~um a n d spinach 6 (Fig. 2 a n d T a b l e I). In c o n t r a s t to this, in families of soluble p r o t e i n s the h y d r o p h i l i c r e s i d u e s are usually less c o n s e r v e d t h a n the h y d r o p h o b i c ones in the protein core [21]. Since C F 1 ~ is a subunit of a p e r i p h e r a l m e m b r a n e p r o t e i n and soluble after isolation, most of its h y d r o p h o b i c r e s i d u e s should be l o c a t e d in the pro-
TABLE I Namber of identities in the primary structure of mature CFl subunit of Spinacea oleracea (187 residues), when compared with Pisum sativum CFI 6 (187 residues) and E. coil F1 ~ (177 residues)
Alignment as in Fig. 2. In brackets: % of identities in relation to all residues within the respective group of amino acids of 6 in the respective species. Comparison ofsubunits 6 from
Total identities
Hydrophilic DETSKR HNQCY
Hydrophobic LVIMFP W
Small AG
Spinach and
106
55 (57%of 96) 18 (22% of 81)
33 (50%of 66) 14 (23%of 61)
18 (72%of 25) 9 (26%of 35)
satil,um
Spinach and E. coli
41
tein core a n d most of the h y d r o p h i l i c r e s i d u e s at the surface. Thus, the surface of ~ is surprisingly well conserved. T h e function of ~ s e e m s to d e p e n d not only on its size a n d shape, b u t on its surface; its specific i n t e r a c t i o n s with o t h e r subunits of the A T P - s y n t h a s e c o m p l e x a r e most i m p o r t a n t . Using a n t i b o d i e s and limited proteolysis we have shown that C F 1 subunit in situ is mostly inaccessible, i n d e e d [22]. In hybrid r e c o n s t i t u t i o n the similarities on the surface of 6 from E. coli a n d S. oleracea w e r e not sufficient for catalytic r e c o n s t i t u t i o n [9]. W e p r o p o s e that the capacity of CFI subunit 6 to r e c o n s t i t u t e p h o s p h o r y l a t i o n activity, corr e l a t e s with the n u m b e r of identical h y d r o p h i l i c surface residues. F o r financial s u p p o r t we are i n d e b t e d to the Studie n s t i f t u n g des D e u t s c h e n V o l k e s (J.A.H.) and the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (Be 664 to R.J.B.).
References 1 Jagendorf, A.T., McCarty, R.E. and Robertson, D. (1991) in The Photosynthetic Apparatus: Molecular Biology and Operation (Bogorad, L. and Vasil, I.K., eds.), pp. 225-254, Academic Press, New York. 2 Smith, J.B. and Sternweis, P.C. (1977) Biochemistry 16, 306-311. 3 Xiao, J. and McCarty, R.E. (1989) Biochim. Biophys. Acta 976, 203-209. 4 Engelbrecht, S. and Junge, W. (1988) Eur. J. Biochem. 172, 213-218. 5 Berzborn, R.J. and Finke, W. (1989a) Z. Naturforsch. 44c, 153 160. 6 Beckers, G. Berzborn, R.J. and Strotmann, H. (1992) Biochim. Biophys. Acta 1101, 97-104. 7 Mitchell, P. (1985) FEBS Lett. 182, 1 7. 8 Boyer, P.D. (1987) Biochemistry 26, 8503-8507. 9 Engelbrecht, S., Deckers-Hebestreit, G., Altendorf, K.H. and Junge, W. (1989) Eur. J. Biochem. 181,485-491 10 Pucheu, N.L. and Berzborn, R.J. (1984) in Advances in Photosynthesis Res. (Sybesma, C., ed.), Vol. II, pp.571-574, M. Nijhoff/Dr. W. Junk Publ., The Hague. 11 Hermans, J., Rother, C., Steppuhn, J. and Herrmann, R.G. (1988) Plant Mol. Biol. 10, 323-330 12 Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Proc, Natl. Acad. Sci. USA 85, 8998-9002. 13 Yanisch-Perron, C., Viera, J. and Messing, J. (1985) Gene 33, 103-119. 14 Sanger, F.~ Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 15 Lien, S. and Racker, E. (1970) Methods Enzymol. 23, 547-555. 16 Lugtenberg, B., Meijers, J., Peters, R., Van der Hoek, P. and Van Alphen, L. (1975) FEBS Lett. 58, 254-258. 17 Hewick, R.M., Hunkapiller, N.W., Hood, L.E. and Dreyer, W.J. (1981) J. Biol. Chem. 256, 7990-7997. 18 Hoesche, J.A. and Berzborn, R.J. (1992) unpublished data. 19 Von Heijne, G., Steppuhn, J. and Herrmann, R.G. (1989) Eur. J. Biochem. 180, 535-545. 20 Walker, J.E., Saraste, M. and Gay, J.E. (1984) Biochim. Biophys. Acta 768, 164-200 21 Schulz, G.E. and Schirmer, R.H. (1979) Principles in protein structure, Springer, New York, p. 170 22 Berzborn, R.J. and Finke, W. (1989b) Z. Naturforsch. 44c, 480486.
